Efficiency of sperm-mediated gene transfer in the ovine by laparoscopic insemination, in vitro fertilization and ICSI

• Autores: Pereyra Bonnet, Federico; Gibbons, Alejandro; Cueto, Marcela Isabel; Sipowicz, Pablo; Fernández Martín, Rafael; Salamone, Daniel Felipe
• Año de publicación: 2011
• Idioma: inglés
• Tipo de recurso: artículo
• Fuente: Journal of Reproduction and Development; Vol.57, no.2; 188-196; http://reproduction.jp/index-j.php
• Materia: GREEN FLUORESCENT PROTEIN; TRANSGENESIS; ARTIFICIAL INSEMINATION; COMPARATIVE STUDY; EMBRYO DEVELOPMENT; FLUORESCENCE IN SITU HYBRIDIZATION; GENE TRANSFER; INTRACYTOPLASMIC SPERM INJECTION; LAPAROSCOPY; POLYMERASE CHAIN REACTION; PRENATAL DEVELOPMENT; SHEEP; TRANSGENIC ANIMAL; ANIMALS; ANIMALS, GENETICALLY MODIFIED; EMBRYONIC DEVELOPMENT; GENE TRANSFER TECHNIQUES; IN SITU HYBRIDIZATION, FLUORESCENCE; INSEMINATION, ARTIFICIAL; SPERM INJECTIONS, INTRACYTOPLASMIC; OVIS; OVIS ARIES
• Institución: Universidad de Buenos Aires. Facultad de Agronomía

Fil: Pereyra Bonnet, Federico. Universidad de Buenos Aires. Facultad de Agronomía. Laboratorio de Biotecnología Animal. Buenos Aires, Argentina.
Fil: Gibbons, Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). EEA Bariloche. Laboratorio de Reproducción de Rumiantes Menores. San Carlos de Bariloche, Río Negro, Argentina.
Fil: Cueto, Marcela Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). EEA Bariloche. Laboratorio de Reproducción de Rumiantes Menores. San Carlos de Bariloche, Río Negro, Argentina.
Fil: Sipowicz, Pablo. Universidad Nacional de General San Martín. Laboratorio de Neuro y Citogénetica Molecular. Buenos Aires, Argentina.
Fil: Fernández Martín, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Laboratorio de Biotecnología Animal. Buenos Aires, Argentina.
Fil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Laboratorio de Biotecnología Animal. Buenos Aires, Argentina.
Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams. The embryos were recovered by flushing the uterine horns. For IVF and ICSI, slaughterhouse oocytes were fertilized with DNA-treated frozen/thawed sperm. All recovered embryos were exposed to blue light (488 nm) to determine green fluorescent morulae and blastocysts rates. High cleavage and morulae/blastocysts rates accompanied the LI and IVF procedures, but no egfp-expressing embryos resulted. In contrast, regardless of the sperm/ plasmid incubation treatment, egfp-expressing morulae and blastocysts were always obtained by ICSI, and the highest transgenesis rate (91.6 percent) was achieved with Short Incubation. In addition, following the incubation of labeled plasmid DNA, after Long or Short exposure treatments, with fresh or frozen/thawed spermatozoa, only non-motile fresh spermatozoa could maintain an attached plasmid after washing procedures. No amplification product could be detected following PCR treatment of LI embryos whose zonae pellucidae (ZP) had been removed. In order to establish conditions for transgenic ICSI in the ovine, we compared three different activation treatments, and over 60 percent of the obtained blastocysts expressed the transgene. For ICSI embryos, FISH analysis found possible signals compatible with integration events. In conclusion, our results show that in the ovine, under the conditions studied, ICSI is the only method capable of producing exogenous gene-expressing embryos using spermatozoa as vectors.